1. Field of the Invention.
The present invention relates to microfabricated recessed disk microelectrodes. More specifically, this invention relates to microcavities containing microelectrodes and being separated from surrounding media by a lipid bilayer that is anchored to the rim of the microcavity. The invention also relates to microcavities having a hole in the bottom in order to relieve osmotic pressure. The invention also relates to arrays of such microcavities.
2. Prior Art
Microelectrode based electrochemical analysis systems are advantageous over systems containing macroelectrodes. First, microelectrodes can be made very small, for example bands with widths 32 nm and single microdisks with diameters of 2 nm. Second, the current density for microelectrodes is greater than that at macroelectrodes due to radial diffusion. This results in a measurable steady state current at electrodes of the dimensions described above. Finally, uncompensated resistance does not induce large potential drops due to the small currents drawn by micro electrodes.
Microelectrodes have been used for analysis in small volumes. Two general approaches have been used for the analysis, differentiated by the construction of the system. The first type of system uses single electrodes placed in solution. The second type of system uses microfabrication techniques to pattern the electrodes. The smallest volumes, 0.6 nL, using this technique have been demonstrated.
Microelectrodes have been used for analysis in convective systems. One example of this is the use of microelectrodes as detectors in liquid chromatography and capillary electrophoresis. The baseline noise, which also helps determine the limit of detection is dependent upon the flow rate (i.e. convection). A number of different geometries have been reported, with the band and disks being among the most common.
Microelectrodes, in general, have been studied extensively and reviews have been published. Microelectrodes have been used in complex media such as blood and urine. They have also been used to provide spatially-resolved information from surfaces and cell membranes. A host of biosensor applications has been reported.
The most commonly studied microelectrode geometry is the disk because it is relatively simple to construct and can attain true steady state current. Both planar (PDM) and recessed disk (RDM) microelectrodes have been studied. A recessed microdisk resides at the bottom of a cavity whose walls are made of insulator material. Although the current measured at RDMs is typically less than that at PDMs of equal radius, it can be independent of convection outside of the cavity, depending upon the cavity's dimensions and the strength of the convective forces. Two components give an RDM its unique properties: the size of the electrode and its position, which is set back from the main plane of the insulating layer of the substrate.
Recessed microdisk electrodes were originally constructed from in-plane microdisk electrodes. Either chemical or electrochemical etching has been used to etch the electrodes away from the surface plane of the insulator. The depth of the cavity and surface roughness of these electrodes are difficult to control. The early applications included chemical measurements in convective systems.
The incorporation of membrane proteins and enzymes into modifying layers on surfaces is of interest for model systems of biomembranes and for the development of chemical sensors. Membrane protein structure and function are highly dependent on the surrounding environment, and thus, it is essential to design materials on surfaces that provide the necessary characteristics to host such proteins. Langmuir-Blodgett (LB) techniques have been used to assemble phospholipids onto surfaces to provide biomembrane-like environments. These assemblies have been characterized by AC impedance measurements, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy. Another more recent method that has the advantage over LB methods of ease of formation involves vesicle fusion to surfaces to form hybrid bilayers. In this method, the chemistry of the strong interaction of sulfur with gold is used to form self-assembled monolayers (SAMs) to produce a first layer. This layer provides the driving force for deposition of a second layer of phospholipids by hydrophobic coupling of phospholipid vesicles to the SAM layer. The first layer is chemisorbed rather than physisorbed and highly ordered, which provides a more ordered foundation for additional multilayer construction. Also in forming the second layer, a water rinse, rather than an organic solvent rinse, flushes away unbound vesicles and lipids without removing biomolecules incorporated within the surface-confined layers.
An area of great importance is the incorporation of biological molecules onto electrodes to aid in electrochemical detection of analytes. An important aspect is the chemistry behind modifying the electrode without destroying the activity of the biological molecule, many chemical methods have been developed to modify electrode surfaces. Immobilizing methods for biological molecules such as enzymes include covalent bonding, adsorption, monolayer deposition, entrapment, and microencapsulation. Currently, SAMs are being studied as immobilization tools because of the ease with which they create highly ordered organic films. A recent example is use of SAMs of alcohol-terminaled alkanethiols and glucose oxidase on gold electrodes to prepare a glucose sensor.
Providing the necessary hydrophobic and hydrophilic properties on the electrode surface is a challenging problem. The native environment for many proteins and enzymes is the cellular biomembrane. Several methods are being developed to artificially recreate that environment on electrode surfaces. Surface-confined lipid membranes on electrodes have been formed with Langmuir-Blodgett (LB) techniques and by combining SAMs with phospholipid vesicles. Controlling access to the underlying surface has been demonstrated using gate sites through a monomolecular LB film.
Bilayer formation using SAMs and phospholipid vesicles has been studied by electrochemistry and surface plasmon resonance properties of hybrid bilayers by cyclic voltammetry using Fe(CN)63− as the redox species in an electrolyte solution of 1 M KCl and determined that the presence of the hybrid bilayer reduces the rate of electron transfer by approximately two orders of magnitude from that of the bare electrode. Plant has also compared capacitance values obtained by impedance measurements of SAMs of alkanethiols to those of hybrid bilayers of octadecanethiol (C18SH) and 1-palmitoyl-2-oleoylphosphhatidylcholine (POPC) and reports that hybrid bilayers are sufficiently flexible to accommodate a molecule such as a pore forming mellitin. A glucose sensor that is based on a similar bilayer-self assembling technique has also been reported. This method involves using tetracyanoquinodimethane (TCNQ) that resides within a dodecanethiolphosphatidylcholine and phosphatidylethanolamine bilayer, and serves as a mediator between the underlying electrode and overlying, cross-linked glucose oxidase. However, the bilayer thickness, estimated from impedance measurements, was smaller than typical values reported in the literature, and the TCNQ diffused out of the bilayer during electrochemical measurements.
Membrane assembly methods have been used in conjunction with enzyme reconstitution procedures to modify the electrode surface and study the electron-transfer reaction of immobilized bovine cytochrome c oxidase. Cyclic voltammetry and potential step chronoabsorptometry were used to show the direct electron transfer between the gold substrates and the cytochrome c oxidase incorporated in a dodecanethiol and 1-palmitoyl-2-oleoylphosphhatidylethanolamine (POPE) and POPC layers. In addition, the immobilized enzyme was shown to both reduce and oxidize the cytochrome c in solution. Others have used vesicles formed from molecules with head groups having a net positive or negative charge such as dimethyldioctadecylammoniumbromide (DODAB) and dimyristoyl phosphatidylglycerol (DMPG), respectively. Lipid bilayers and trilayers on solid supports can be formed by fusing these charged vesicles to a charged monolayer such as carboxylate mercaptans directly or via a cation linkage. These layers have been analyzed by impedance and surface plasmon resonance to determine the mean thickness of the membranes. Impedance analysis combined with spectroscopy has provided discrimination in identifying between specific and non-specific adsorption of streptavidin and biotinated-lipids.
Techniques to create supported bilayers using vesicles that form a top fluid layer of phospholipids onto a fixed SAM have also been demonstrated. Permeation of ions through these bilayers was studied upon incorporating the pore-forming peptide mellitin. A glucose sensor that is based on a similar bilayer-self assembling technique has also been reported. The assembly involved tetracyanoquinodimethane which resides within an alkanethiol/phospholipid bilayer, and serves as a mediator between the underlying electrode and overlying, cross-linked glucose oxidase.
Because of its simple composition and characteristic function dependence on structure, Gramicidin A is used as a convenient probe to evaluate modifying layers on electrodes. This small ion channel-forming peptide is one of the best characterized and most extensively studied membrane polypeptides. It is an antibiotic that is isolated from Bacillus brevis and is active against Gram-positive bacteria. It consists of an alternating L, D pentadecapeptide with the primary sequence of HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-(L-Trp-D-Trp)3-L-Trp-NHCH2CH2OH. The 3-dimensional conformation of gA is complex and dependent upon its environment. In biological or model membrane systems, gA adopts an ion channel conformation which allows the passage of water and small, monovalent cations. The channel is in the form of two β-helical monomers that dimerize end to end with the formyl-NH ends associated in the center of a lipid bilayer. The length of the dimer is approximately 26 Å. The peptide backbone forms a hydrophilic pore that has a diameter of about 4 Å. Gramicidin A has been characterized in only a few electrode-modified systems. Evidence has been presented of the selectivity of gA toward metal mono cations on electrode surfaces. Gramicidin was incorporated into dioleoyl phosphatidylcholine and bovine brain phosphatidylserine monolayers using LB techniques on mercury drop electrodes. By cyclic voltammetry, selective permeability to TI+ over Cd2+ for layers containing gA is consistent with the gA being in the ion channel conformation. This system is not easily conducive to further evaluations by spectroscopy due to the nature of mercury. Different preparation techniques for supported lipid layers have been evaluated by impedance analysis. Gramicidin has been incorporated into one type of film, a SAM of 3-mercaptopriopionic acid, covered with a bilayer of DODAB, formed from fusion of DODAB vesicles, to create trilayer films. Again, spectroscopic characterization was not performed. However, electrochemical behavior was observed in the presence of Cs+ and Sr2+ that might be interpreted as gramicidin channels controlling ion permeation through the film.
It is therefore desirable to produce both tubular nanoband and recessed disk microelectrodes within a microcavity capable of detecting electrical currents undistorted by convection of a solution.
It is also desirable to produce microcavities having microelectrodes and a lipid bilayer extending across the top of the microcavity.
It is also desirable to produce microcavities having microelectrodes, a lipid bilayer, and a hole in the bottom to reduce osmotic effects.
It is also desirable to produce arrays of microcavities.
It is also desirable to develop an accurate, efficient and reproducible method for creating microcavities or arrays thereof having microelectrodes, lipid bilayers and holes to reduce osmotic effects.